5-[2-(N-(Substituted phenyl)acetamide)]amino-1,3,4-thiadiazole-2-sulfonamides as Selective Carbonic Anhydrase II Inhibitors with Neuroprotective Effects

Article abstract of DOI:10.1002/cmdc.201900703

In this study, 22 novel compounds were designed and synthesized by acetamide bridge chains, among which 5 a–5 k were monosubstituted compounds, and 6 a–6 k were disubstituted. A series of biological evaluations was then carried out to determine the carbonic anhydrase inhibitory activity, neuroprotective effects and cytotoxicity of 5 a–5 k and 6 a–6 k. The results showed that some compounds could protect PC12 cells from sodium nitroprusside (SNP)-induced damage. In terms of the neuroprotection and inhibitory activity against carbonic anhydrase II, monosubstituted compounds were better than disubstituted. Compound 5 c exhibited better protective effect in PC12 cells than that of edaravone, and 5 c also showed less cytotoxicity. In addition, compound 5 c was found to be the most effective selective carbonic anhydrase II inhibitor (IC₅₀=16.7 nM, CAI/CAII=54.3), which was similar to the inhibitory effect of acetazolamide. Moreover, the selectivity of compound 5 c was better than that of acetazolamide (IC₅₀=12.0 nM, CAI/CAII=20.8). Molecular docking presented that the binding effect of compound 5 c with carbonic anhydrase II was superior to that of 5 c with carbonic anhydrase I and IX, which was consistent with the inhibitory results. Based on above findings, compound 5 c may be a potential candidate for selective carbonic anhydrase II inhibitor, and it had obviously neuroprotective effect and great advantages in drug safety.

Full text of DOI:10.1002/cmdc.201900703

Accepted Article
Title: 5-[2-(N-(substituted phenyl) acetamide)] amino-1, 3, 4-thiadiazole
-2-sulfonamides as selective carbonic anhydrase II inhibitor with
neuroprotective effects
Authors: jingxia zhang, Caibao Jiang, Jinguo Shi, Liping Liao, Liantao
Zhang, Jiayong Liu, Yang Wang, and Yaoqiang Lao
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201900703
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900703
A Journal of
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5-[2-(N-(substituted phenyl) acetamide)] amino-1,3,4-thiadiazole-2-sulfonamides as selective carbonic anhydrase II
inhibitor with neuroprotective effects
Caibao Jiang+,[a] Jinguo Shi+,[a] Liping Liao, [a] Liantao Zhang,[a] Jiayong Liu,[a] Yang Wang,[a] Yaoqiang Lao,[a] Jingxia Zhang*[a]
[a]
Dr. C.B. Jiang,+ J. G. Shi,+ L. P. Liao, L. T. Zhang, J. Y. Liu, Y. Wang, Y. Q. Lao, Prof. J. X. Zhang
Department of Medicinal Chemistry, School of Pharmaceutical Science, Sun Yat-sen University, Guangzhou, 510006, P. R. China
E-mail: zhjingx@mail.sysu.edu.cn
+ These authors contributed equally to this work.
Abstract: In this study, 22 novel compounds were designed
and synthesized by acetamide bridge chains, among which
5a-5k were mono-substituted compounds, and 6a-6k were di-
substituted. A series of biological evaluations was then carried
out to determine the carbonic anhydrase inhibitory activity,
neuroprotective effects and cytotoxicity of 5a-5k and 6a-6k.
The results showed that some compounds could protect PC12
cells from sodium nitroprusside (SNP)-induced damage. In
terms of the neuroprotection and inhibitory activity against
carbonic anhydrase II, mono-substituted compounds were
better than di-substituted. Compound 5c exhibited better
protective effect in PC12 cells than that of edaravone and 5c
also showed less cytotoxicity. In addition, compound 5c was
the most effective selective carbonic anhydrase II inhibitor
(IC50=16.7 nM, CAI/CAII=54.3), which was comparable to the
inhibitory effect of acetazolamide. Moreover, the selectivity of
compound 5c was better than that of acetazolamide
(IC50=12.0 nM, CAI/CAII=20.8). Molecular docking presented
that the binding effect of compound 5c with carbonic
anhydrase II was superior to that of 5c with carbonic
anhydrase I and IX, which was consistent with the inhibitory
results. Based on above findings, compound 5c may be a
potential candidate for selective carbonic anhydrase II
inhibitor, and it had obviously neuroprotective effect and great
advantages in drug safety.
far, but only 12 of them are catalytically active.[10-12] CA II is
the most widely expressed isoform and is identified in the
anterior chamber of the eye, and that is also responsible for
the production of bicarbonate and the main constituent of the
aqueous humour.[13] CAI and II are ubiquitous CA isoforms,
and both exist in the cytoplasm. CAII is associated with
epilepsy,[14] glaucoma[15] and neurodegenerative disease,[16]
while CAI is responsible for multiple side effects of CA
inhibitors.[17,18] Therefore, compared with the off-target CA I
isoform, it would have made more sense the evaluation of
hCA II selectivity. hCA IX is a membrane-bound CAs that is
overexpressed in hypoxic tumor cells and associated with
many types of human cancers, which is widely used for
screening tumor targets.[19]
The sulfonamide inhibitors can inhibit all human CA
isoforms. However, they are not safe and limited due to their
non-specificity and side effects, such as gastrointestinal
disorders, acidosis, myopia, depression and sleepiness.[17, 20,
21]
Therefore, it was a key task to develop a novel CA II
inhibitor with specific selectivity and minimal side effects.
In fact, glaucoma is a degenerative optic nerve disease.
And higher intraocular pressure could damage the optic
nerve and cause its function loss. Recently, many
neuroprotectants have been used for treatment of glaucoma
by the means of lowering the intraocular pressure to improve
the nerve function.[22-25] Hence, neuroprotection has become
an important method for the management of glaucoma.
In this study, our aim was to discover a selective
carbonic anhydrase II inhibitor with neuroprotective effects.
The research firstly used acetazolamide as the lead
compound to design and synthesize 22 novel compounds
through acetamide bridge chains. Then it examined the
inhibitory activity against carbonic anhydrase isoforms I, II
and IX, neuroprotective effects on PC12 and the cytotoxicity
of compounds 5a-5k and 6a-6k . At last, the research also
performed docking and ADME properties prediction
Introduction
Glaucoma is one of the main causes of irreversible blindness
in the world.[1, 2] By 2040, the number of glaucoma patients
worldwide is predicted to reach 111.8 million.[3, 4] The clinically
used drugs for the management of glaucoma are carbonic
anhydrase (CA) inhibitors such as acetazolamide,
methazolamide, and ethoxzolamide.[5-7]
CAs is widespread in organisms, varying with respect to
tissue distribution and subcellular localization.[8, 9] Fifteen α-
isozymes (CA I–XV) have been isolated and characterized so
1
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experiments on optimal compounds to find potential
candidates.
slower the reaction proceed. At 25℃, although reaction was
completed in 2 h, only 3% mono-substituted product was
produced. At 0 ℃ , the reaction obtained 5% of mono-
substituted products and the remaining 59% of raw materials.
At -20℃, it obtained 21% of mono-substituted products and
the remaining 56% of raw materials, and no products were
formed at -40℃. As the data of the reaction shown, the
Results and Discussion
Chemistry
Optimization of reaction conditions
According to previous literature,[26, 27] mono- or di-substituted
products cannot be obtained simultaneously in a reaction.
After optimizing the reaction conditions by the study, mono-
and di-substituted compounds were obtained in a reaction.
The synthetic route was shown in Scheme 1. First, 5-amino-
1,3,4-thiadiazole-2-sulfonamide (2) and 2-bromo-N-(4-
fluorophenyl) acetamide (4c) served as reactants, with DMF
reaction would control the temperature at -20 ℃ and
simultaneously control other conditions to maximize
production of mono-substituted and di-substituted products
(Table 1, entries1-4).
(ⅱ) Equivalent ratios of 4c:2: Reactions were carried out
with 1 eq K2CO3 and the equivalent ratios of 4c:2 were 1:1,
1:3 and 1:5 at -20℃. Results discovered that the ratio of
mono- and di-substituted products increased with the
equivalence ratio of 4c:2 increasing. When the equivalent
ratio of 4c:2 was changed to 1:5, raw materials were
29]
as solvent,[28,
and reaction was carried out at room
temperature. Results discovered that only di-substituted
products were formed. Thus, the reaction conditions were
optimized by controlling the reaction temperature, equivalent
ratio of substrates, type and amount of alkali to improve the
yield of mono-substituted product. The analysis was
performed by using HPLC (Fig.1).
completely transformed. As
a result, mono-substituted
products have increased, which accounted for 22% of mono-
and di-substituted products (Table 1, entries 4-6).
( ⅲ ) Types of alkali: Compared with the reaction of
catalyst KHCO3, Na2CO3, CH3COONa or no alkali, the
reaction of catalyst K2CO3 acquired higher ratio of mono-
substituted and di-substituted products, what's more, it was
carried out completely. Therefore, K2CO3 was the optimal
catalyst (Table 1, entries 6-10).
(ⅳ) Alkali dosage: Reactions were carried out at -20℃,
with 1 eq, 0.5 eq, 0.3 eq of K2CO3, and 4c:2 of 1:5. The
amount of K2CO3 was 0.3 eq and 0.5 eq, the reaction retained
69% and 17% of the raw materials respectively. When the
amount of K2CO3 was 1eq, the reaction went on completely.
Meanwhile, the reaction rate of 1 eq K2CO3 was high than that
of 0.3 eq and 0.5 eq (Table 1, entries 6, 11-12).
Based on above experimental datas, the optimal
reactions were carried out at -20℃, with 1 eq of K2CO3, and
4c:2 of 1:5.
Fig.1 HPLC of 4c, 5c and 6c (A: 4c+5c+6c, B: Reaction
solution)
(ⅰ) Temperature: Reactions were carried out at 25, 0, -
20 and -40℃, with 1 eq K2CO3 and the equivalent ratio of 4c:2
of 1:1. Results found that the lower the temperature was, the
2
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Fig.2 1HNMR of (A) 5c and (B) 6c
Scheme 1. Reaction scheme for synthesis of compounds 5a-
5k and 6a-6k. Reagents and conditions: ( ⅰ ) 3mol/L
Hydrochloric acid, reflux, 3h; (ⅱ) Glacial acetic acid, 5%
sodium acetate, bromoacetyl bromide, room temperature, 1 h;
(ⅲ) Dimethyl sulfoxide, potassium carbonate, 4h.
Biological tests
Carbonic anhydrase inhibition
The values of inhibition by compounds 5a-5k and 6a-6k
against hCA I, II and IX were determined by an esterase
assay[30-31]. Results are shown in Table 2.
( ⅰ ) Compounds 5a-5k and 6a-6k had moderate
inhibitory activity against hCA I. IC50 values of compounds 5a-
5k ranged from 907.3 to 2683.0 nM, and those of compounds
6a-6k ranged from 2664 to 7080 nM.
Table 1. Optimization of reaction conditions
Entry
Base
Alkali
(eq)
4c/2
T (℃)
5c/6c
(5c+6c)%
1
1
1/1
1/1
1/1
1/1
1/3
1/5
1/5
1/5
1/5
1/1
1/5
1/5
25
3/97
5/95
21/79
/
100
41
44
/
(ⅱ) Compounds 5a-5k had significant inhibitory effect
against hCA II which IC50 values of compounds 5a-5k ranged
from 16.7 to 516.3 nM, in contrast, most of compounds 6a-6k
exhibited moderate-weak inhibitory activity which compounds
6a-6k ranged from 45.9 to 4698 nM. Compounds 5c (F-
substituted product) was the most active, with an IC50 of 16.7
nM.
1
2
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
0
1
-20℃
-40℃
-20℃
-20℃
-20℃
-20℃
-20℃
25
3
1
4
1
15/85
22/78
22/78
13/87
/
97
100
51
79
/
5
1
6
K2CO3
KHCO3
1
(ⅲ) Different phenyl ring substituents played a key role in
inhibitory activity against CA II. IC50 values of compounds 5a,
5c, 5h, 5i, 5j and 5k were less than 100 nM. For F-substituted
products, para-F product was strongest (para-F (5c) > meta-
F (5j) > ortho-F (5k), with IC50 values of 16.7 nM, 54.7 nM, and
73.4 nM, respectively). When the halogen atom was at para-
position of benzene ring, 4-F product was the most effective
(4-F (5c) >4-Cl (5a) >4-Br (5d) > 4-I (5g), with IC50 values of
16.7 nM, 90.6 nM, 212.8 nM, and 516.3 nM, respectively).
(ⅳ) For compound 5c, IC50 values against hCA I and hCA
II were 907.3 nM and 16.7 nM, and the selectivity of hCA I/CA
II was 54.3. Otherwise, for acetazolamide as a positive control
drug, IC50 values against hCA I and hCA II were 250.0 nM and
12.0 nM, and the selectivity of hCA I/CA II was 20.8.
Compound 5c had an IC50 of 16.7 nM for CA II, which was
comparable to the inhibitory effect of acetazolamide, but the
selectivity of 5c against hCA I/CA II was 2.6 times as strong
as that of AZA. Therefore, 5c may have fewer side effects
than acetazolamide.
7
Na2CO3
CH3COONa
no base
1
8
1
9
1
/
/
10
11
12
0.5
0.3
13/87
4/96
83
31
-20℃
-20℃
K2CO3
K2CO3
Characterization of structures
The structures of target products were verified by NMR, MS,
UV and IR. The purity of these compounds was determined
by HPLC. The difference between two series can be detected
with 1HNMR, for example, mono-substituted product (5c) had
N-H signals with 1HNMR (Fig. 2A), while di-substituted
product (6c) had not (Fig. 2B).
3
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(v) Compounds 5a-5k and 6a-6k had weak inhibitory
activity against hCA IX. Except for compounds 5a, 5e, 5g, 5h
and 5i, the IC50 values of rest compounds were all more than
10000 nM. Acetazolamide, a broad and non-selective CA
inhibitor, had an IC50 of 25 nM for CAIX. The difference was
that compound 5c had poor selectivity for CAIX and it only
targeted CAII.
Table 2. Inhibitory activity of target compounds on carbonic anhydrase I, II and IX
Entry
R
hCAI
hCA II
hCAIX
(IC50, nM)1,2
(IC50, nM)1,2
(IC50, nM)1,2
Comps 5
Comps 6
3964.0
7080.0
2664.0
4276.0
4661.0
5781.0
4886.0
3861.0
4130.0
2789.0
34480.
Comps 5
Comps 6
392.7
1100.0
45.9
Comps 5
Comps 6
＞10000
＞10000
＞10000
＞10000
＞10000
＞10000
＞10000
＞10000
＞10000
＞10000
＞10000
a
4-Cl
4-OCH3
4-F
1611.0
2060.0
907.3
90.6
305.0
16.7
＞10000
＞10000
4757
b
c
f
4-Br
1492.0
1543.0
1123.0
2683.0
1214.0
1188.0
1058.0
1244.0
212.8
309.5
221.6
516.3
74.2
732.9
926.8
730.2
4698.0
157.4
629.8
89.3
＞10000
4499
e
4-CF3
4-CH3
4-I
f
＞10000
2300
g
h
4-C(CH3)3
4-H
5471
i
209.8
54.7
2285
j
k
3-F
＞10000
＞10000
2-F
73.4
178.2
AZA3
250.0
12.0
25.0
1 IC50 values were determined by the 4-nitrophenyl acetate (4-NPA) esterase assay.
2 Mean from 3 different assays, by an esterase assay (errors were in the range of ± 5-10% of the reported values).
3 Acetazolamide as positive reference material.
Compounds 5a-5k and 6a-6k were preliminarily
Cell viability assay
screened by MTT assay. Edaravone was used as a positive
The excessive release of NO can cause neurotoxicity and
control (edaravone is known as a free radical scavenger and
many neurodegenerative diseases[32,
by mediating
33]
is used to treat stroke in Japan).[36] Preliminary screening
result (Fig. 3B) displayed that compounds 6a-6k not only
exhibited less protective effects, but also produced
cytotoxicity. While compounds 5a, 5b, 5c, 5g and 5i could
effectively protect PC12 cells, and their activities were better
than that of edaravone.
oxidative stress in human body. SNP is widely used as a direct
donor of NO in experiments.[34, 35] Rat pheochromo cytoma
PC12 cells are commonly used as nerve cells in study of
neurodegenerative diseases.[36] First, SNP-induced PC12 cell
injury model was established. The PC12 cells were cultured
with SNP at different concentrations ranging from 500 to 700
μM for 24 h, and SNP concentration which could result in cell
viability up to 60% was obtained. As shown in Fig. 3A, SNP
concentration of 650 μM was chosen.
The protective efficacy of different concentrations of
compounds 5a, 5b, 5c, 5g and 5i was assessed to further
investigate and compare their potency by MTT assay. As
shown in Fig. 3C, compounds 5a, 5b, 5c, 5g and 5i exhibited
dose-dependent protective effects. The protective effects of
4
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compounds 5a, 5b, 5c, 5g and 5i increased significantly at 20
μM better than 50 μM edaravone and 20 μM acetazolamide.
to all three cell lines and showed a great advantage in safety
over positive control acetazolamide.
Table 3. Cytotoxic activity of compounds 5a-5k against
HEK293, LO2 and PC12 cell lines
HEK293
IC50 (μM)
139.2±3.3
LO2
PC12
Comps
R
IC50(μM)
183.8±4.5
IC50 (μM)
4-Cl
4-OCH3
4-F
＞200
＞200
＞200
＞200
＞200
＞200
＞200
＞200
＞200
＞200
＞200
＞200
5a
5b
5c
5d
5e
5f
140.3±1.7
157.5±0.2
144.8±4.7
136.1±2.7
137.9±3.5
143.9±4.4
1575±2.8
163.5±5.5
156.7±1.6
152.6±3.6
144.7±19.9
223.0±5.4
243.1±7.5
216.5±4.9
198.3±4.8
176.2±6.5
168.8±6.0
162.4±4.7
221.8±3.45
228.0±1.8
188.8±2.4
169.3±3.3
4-Br
4-CF3
4-CH3
4-I
5g
5h
5i
4-C(CH3)3
4-H
Fig. 3 PC12 cell viability (A) PC12 cells were treated with
different doses of SNP followed by incubation for 24 h. (B)
PC12 cells were pretreated with 50 μM edaravone and 20 μM
target compounds and AZA for 2 h followed by treatment with
650 μM SNP for another 24 h. (C) PC12 cells were pretreated
with 5, 10, 20 μM 5a, 5b, 5c, 5g, 5i and 25, 50 μΜ edaravone
for 24 h followed by treatment with 650 μM SNP for another
24 h and measured by MTT assay. (Data were expressed as
the mean ± SD of three replicates ***p < 0.0005, **p < 0.005
and *p < 0.05 compared to SNP group).
3-F
5j
2-F
5k
AZA
Computational studies
Molecular docking
Molecular docking studies were conducted to better
understand the interactions between ligands and receptors.
Discovery Studio 2016 client was used to dock compound 5c
Cytotoxicity assay
with CAI protein 5WLU,[37] CAII protein 5ULN,[38] and CAIX
The cytotoxicity of compounds was an important index for
evaluation of medicinal properties in drug research and
development. The cytotoxicity of compounds 5a-5k against
HEK293 (human embryonic kidney normal cell line), LO2
(human normal hepatocytes) and PC12 (mouse pheochromo
cytoma) cells was determined by MTT method in vitro (Table
3). In HEK293 cells, IC50 values of 5a-5k ranged from 136.10
± 2.72 μM to 163.50 ± 5.50 μM, which were all above 100 μM
and higher than that of acetazolamide (IC50=135.20 ± 1.74
μM). Thus, compounds 5a-5k could be considered essentially
non-toxic to HEK293 cells. For LO2 cells, IC50 values of 5a-5k
ranged from 162.40 ± 4.68 μM to 243.10 ± 7.53 μM, all of
which were more than 100 μM. IC50 values of the rest
compounds were higher than that of acetazolamide
(IC50=169.30 ± 3.33 μM), except 5g and 5h. For PC12 cells,
IC50 values of 5a-5k and acetazolamide were more than 200
μM. Therefore, compounds 5a-5k were considered non-toxic
[39]
protein 5JN3
(all from the RCSB PDB data bank), and
conformation with the highest binding energy was selected for
further analysis. The binding modes of compound 5c with
5WLU, 5ULN, 5JN3 were observed by PyMol. His94, His96,
His119 coordinate with Zn2+ to form all hCAs catalytic sites.
The binding mode of compound 5c in active site of CA I was
stabilized by the four H-bonds interaction with Thr199, Thr200,
Gln67, Ser65, and by the coordination of sulfonamide and zinc
ion through negatively charged nitrogen (Fig. 4A-B). The
active site residues Leu57, Ser73 were involved in
hydrophobic interaction (Fig. 4A-B). Docking results of
compound 5c and CAII exhibited that compound 5c interacted
stably with Thr199, Leu198 and Asn67 by four H-bonds in the
active site of CAII. And sulfonamide, through negatively
charged nitrogen, coordinated to zinc ion, which also
contributed to the stability (Fig. 4C-D). At the same time,
5
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residue Phe131 interacted stably with benzene ring through
π-π interaction. The active site residues Trp245, Phe131 and
Asn62 participated in hydrophobic interactions. (Fig. 4C-D). At
the active site of CA IX, compound 5c formed three H-bonds
interaction with Thr199 and His64, whereas residues Tyr7,
Asn62, and Leu60 were involved in hydrophobic interaction
(Fig. 4E-F).
In Figures 4C-D, the optimal position for compound 5c
binding to CAII was presented and demonstrated the ability to
form an interaction network within the depths of nearby zinc
ion catalytic sites. The sulfonamide group was one of the most
vital functional groups in carbonic anhydrase inhibitors. The
sulfonamide group of 5c was in polar contact with the cavity
of CAII. At the same time, 5c formed hydrophobic interactions
and factional interactions with multiple amino acids of CAII.
Although 5c also formed a hydrophobic interaction with the
amino acids of CAI and IX, the sulfonamide group of 5c was
not strong enough to bind with CAI and IX cavities. This may
explain why the inhibitory activity of 5c on CAII was better than
that of CAI and IX. Molecular docking results were consistent
with those determined by an esterase method.[37-39] In addition,
molecular docking experiment supported compound 5c could
become a new selective carbonic anhydrase II inhibitor.
Fig. 4 Molecular Docking studies of compound 5c with
CAI, CAII and CAIX: Cartoon view of (A) compound 5c
docked with CA I. (B) CAI active site residue interacted with
compound 5c. (C) compound 5c docked with CA II. (D) CAII
active site residue interacted with compound 5c. (E)
compound 5c docked with CA IX. (F) CAIX active site residue
interacted with compound 5c.
Residues are shown with ball and stick and compound 5c was
shown with stick model. Hydrogen bonds were shown as
broken lines (black).
ADME properties
ADME properties of drugs such as absorption, distribution,
metabolism and excretion were the very important indicators
for drug formulation. ADME properties of compound 5c and
positive control drug AZA were predicted by Molinspiration
online calculation tool. Study of ADME can help researchers
better understand properties of drugs. According to Table 4,
compound 5c exhibited good ADME properties, that met the
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requirements with respect to TPSA, MW, miLogP, n-ON
acceptors and n-OHNH donors. The results supported that
compound 5c was more likely to be a candidate for future
drugs.
Table 4 The importance of pharmacokinetic parameters for good oral bioavailability of synthesized compound
Entry
%ABS
MW
MiLogP
TPSA(A2)
n-ROTB
MV
n-ON
acceptors
n-OHNH
donors
Nviolations
≤500
≤5
—
—
≤1
＜140
＜10
＜5
Rule
5c
—
331.4
0.8
127.1
5
246.1
8
4
0
65.2
222.3
-1.2
115.1
2
157.1
7
3
0
AZA
69.3
MW: molecular weight, miLog P: logarithm of partition coefficient of compound between n-octanol and water, TPSA:
topological polar surface area, n-ROTB: number of rotatable bonds, MV: molecular volume, n-ON acceptors: number of
hydrogen bond acceptors, n-OHNH donors: number of hydrogen bonds donors.
Anhydrous solvents, and all of the amines were obtained
Conclusion
from Aladdin or Macklin. 4-Nitrophenyl acetate (4-NPA),
The research synthesized a series of acetazolamide
HEPES, Brij and sodium chloride were all obtained from
derivatives, mono-substituted compounds 5a-5k and di-
Aladdin. Buffer solution (Brij) containing 4-(2-hydroxyethyl)-
substituted compounds 6a-6k. The inhibitory activities of all
1-piperazineethanesulfonic acid (HEPES, 15 mmol/L,
compounds on human carbonic anhydrases I, II and IX were
pH=7.4) and tetraethylene glycol monododecyl ether
studied. Mono-substituted compounds 5a-5k were superior
(0.01%) and NaCl (100 mmol/L) was utilized. hCA I was
to di-substituted compounds 6a-6k in biological evaluation.
purchased from R&D Systems Inc. hCA II and IX were
Compounds 5a-5k displayed significant selective inhibition
purchased from Sino Biological Inc. NMR spectra were
of CA II, and 5c was the best (IC50= 16.7 nM), which was
measured by 500 or 400 MHz nuclear magnetic resonance
comparable to the inhibitory effect of acetazolamide. What’s
(Bruker). Mass spectrometry information was determined by
more, the selectivity for hCA I against hCA II, 54.3, was
a Thermo mass spectrometer. All of UV spectra were
superior to that of lead compound AZA. In addition,
determined by an ultraviolet spectrophotometer (Shimadzu
compounds 5a, 5b, 5c, 5g and 5i showed the better
2600). Thin layer chromatography (TLC) was carried out on
neuroprotection compared with the edaravone in the SNP-
GF254 silica gel (Sijia Biochemical Plastics Factory).
induced PC12 model. And compounds 5a-5k had weaker
cytotoxicity activity against HEK293, LO2 and PC12.
General procedure for the synthesis of 5-amino-1,3,4-
Molecular docking presented that the binding effect of
thiadiazole-2-sulfonamide (2)
compound 5c with carbonic anhydrase II was superior to
carbonic anhydrase I and IX, which was consistent with the
inhibitory results. Besides, ADME properties of 5c indicated
that 5c may have medicine properties. In conclusion,
compound 5c may be the potential candidate drug for novel
selective carbonic anhydrase II inhibitor with neuroprotective
ability.
Acetazolamide (3.00 g, 13.5 mmol) was added to a 250 mL
round bottom flask containing hydrochloric acid (3 M, 23.4
mL) and refluxed at 110 ℃ for 3 h. Mixture was neutralized
with NaOH (4 M, 17.6 mL) and extracted with EtOAc (3 x 200
mL). The combined organic layers were then washed with
brine (1 x 200 mL) and concentrated in a vacuum to obtain
5-amino-1,3,4-thiadiazole-2-sulfonamide. White solid; Yield
60%; 1H NMR (400 MHz, DMSO-d6) δ 8.05 (s, 2H, -
SO2NH2),7.80(s, 2H, -NH2); 13C NMR (101 MHz, DMSO-d6)
δ 172.32, 158.42.
Experimental Section
Chemistry.
Acetazolamide (AZA) was purchased from Macklin.
Bromoacetyl bromide was purchased from Aladdin.
General procedure for the synthesis of 4a-4k
7
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The proper substituted anilines (30 mmol) was added to 12
mL of saturated sodium acetate solution and then 12 mL
acetic acid solution was added to form a suspension.
Suspension was placed in an ice bath, and bromoacetyl
bromide (30 mmol) was added dropwise when the
temperature was less than 5 ℃. After addition, mixture was
removed from the ice bath and stirred for 2 h at the room
temperature. The precipitates were filtered, washed with
distilled water and dried in a vacuum to obtain crude products
4a-4k, which were recrystallized from anhydrous
ethanol.[40,41]
2-bromo-N-(p-tolyl)acetamide (4f): White solid; Yield 90%;
1H NMR (400 MHz, Chloroform-d) δ 8.14 (s, 1H), 7.50-7.37
(m, 2H), 7.18 (d, J = 8.1 Hz, 2H), 4.03 (s, 2H), 2.35 (s, 3H);
13C NMR (101 MHz, Chloroform-d) δ 163.33, 134.98, 134.37,
129.62, 120.20, 29.57, 20.92.
2-bromo-N-(4-iodophenyl)acetamide (4g): White solid;
Yield 85%; 1H NMR (500 MHz, Chloroform-d) δ 8.10 (s,
1H), 7.83-7.57 (m, 2H), 7.48-7.27 (m, 2H), 4.01 (s, 2H); 13
NMR (126 MHz, Chloroform-d) δ 163.36, 138.11, 136.72,
121.79, 88.68, 29.38.
C
2-bromo-N-(4-chlorophenyl) acetamide (4a): White solid;
Yield 90%; 1H NMR (400 MHz, Chloroform-d) δ 8.05 (s, 1H),
7.46-7.38 (m, 2H), 7.29-7.18 (m, 2H), 3.95 (s, 2H); 13C NMR
(101 MHz, Chloroform-d) δ 163.37, 135.49, 130.35, 129.19,
121.28, 29.35.
2-bromo-N-(4-(tert-butyl)phenyl)acetamide (4h): White
solid; Yield 87%; 1H NMR (400 MHz, Chloroform-d) δ 8.16 (s,
1H), 7.44 (d, J = 8.8 Hz, 2H), 7.42-7.30 (m, 2H), 4.00 (s, 2H),
1.30 (s, 9H); 13C NMR (101 MHz, Chloroform-d) δ 163.46,
148.32, 134.29, 125.97, 119.99, 34.48, 31.35, 29.56.
2-bromo-N-(4-methoxyphenyl)acetamide (4b): White
solid; Yield 89%; 1H NMR (400 MHz, Chloroform-d) δ 8.08 (s,
1H), 7.55-7.37 (m, 2H), 7.03-6.79 (m, 2H),4.04 (s, 2H),3.82
(s, 3H); 13C NMR (101 MHz, Chloroform-d) δ 163.30, 157.10,
129.97, 122.05, 114.28, 55.50, 29.49.
2-bromo-N-phenylacetamide (4i): White solid; Yield 90%;
1H NMR (400 MHz, Chloroform-d) δ 8.37-8.06 (m, 1H), 7.78-
7.46 (m, 2H), 7.36 (t, J = 8.0 Hz, 2H), 7.24-7.08 (m, 1H), 4.02
(s, 2H); 13C NMR (101 MHz, Chloroform-d) δ 163.44, 136.92,
129.15, 125.26, 120.09, 29.53.
2-bromo-N-(4-fluorophenyl)acetamide (4c): White solid;
Yield 89%; 1H NMR (400 MHz, Chloroform-d) δ 8.16 (s, 1H),
7.52 (dd, J = 9.0, 4.6 Hz, 2H), 7.07 (t, J = 8.6 Hz, 2H), 4.04
(s, 2H); 13C NMR (101 MHz, Chloroform-d) δ 163.45, 159.92
(d, J = 244.9 Hz), 132.90 (d, J = 2.7 Hz), 122.04 (d, J = 8.1
Hz), 115.87 (d, J = 22.7 Hz), 29.39.
2-bromo-N-(3-fluorophenyl)acetamide (4j): White solid;
Yield 83%; 1H NMR (400 MHz, Chloroform-d) δ 8.30 (s, 1H),
7.48 (dt, J = 10.6, 2.3 Hz, 1H), 7.42-7.26 (m, 1H), 7.18 (ddd,
J = 8.1, 2.0, 0.9 Hz, 1H), 6.86 (tdd, J = 8.3, 2.5, 1.0 Hz, 1H),
4.01 (s, 2H); 13C NMR (101 MHz, Chloroform-d) δ163.76,
162.95 (d, J = 229.3 Hz), 138.43 (d, J = 10.8 Hz), 130.26 (d,
J = 9.3 Hz), 115.36 (d, J = 2.9 Hz), 112.00 (d, J = 21.3 Hz),
107.60 (d, J = 26.4 Hz), 29.33.
2-bromo-N-(4-bromophenyl)acetamide (4d): White solid;
Yield 85%; 1H NMR (400 MHz, Chloroform-d) δ 8.07 (s, 1H),
7.45-7.33 (m, 4H), 3.95 (s, 2H); 13C NMR (101 MHz,
Chloroform-d) δ 163.30, 136.00, 132.15, 121.54, 117.98,
29.36.
2-bromo-N-(2-fluorophenyl)acetamide (4k): White solid;
Yield 85%; 1H NMR (400 MHz, Chloroform-d) δ 8.41 (s, 1H),
8.25 (tt, J = 7.8, 1.2 Hz, 1H), 7.22-7.03 (m, 3H), 4.05 (s, 2H);
13C NMR (101 MHz, Chloroform-d) δ 163.48, 152.73 (d, J =
244.4 Hz), 125.57 (d, J = 10.2 Hz), 125.35 (d, J = 7.7 Hz),
124.65 (d, J = 3.8 Hz), 121.57, 115.03 (d, J = 19.0 Hz), 29.31.
2-bromo-N-(4-(trifluoromethyl)phenyl)acetamide
(4e):
White solid; Yield 88%; 1H NMR (400 MHz, Chloroform-d) δ
8.27 (s, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.6 Hz, 2H),
4.04 (s, 2H); 13C NMR (101 MHz, Chloroform-d) δ 163.59,
139.94, 127.07(q, J = 33.33 Hz), 126.42(q, J = 4.0 Hz),
124.46(q, J = 272.7 Hz), 119.62, 29.26.
General procedure for synthesis of 5a-5k and 6a-6k
First, the mixture of 5-amino-1,3,4-thiadiazole-2-sulfonamide
2 (20 mmol) and potassium carbonate (4 mmol) was added
8
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to a 100 mL round-bottom flask, and 20 mL of DMF was
added to form a suspension. After 5-amino-1,3,4-thiadiazole-
2-sulfonamide 2 was completely dissolved, the suspension
was cooled to -20 ℃ and stirred for 5 minutes before adding
benzoyl bromides 4a-4k. Reaction progress was monitored
by TLC. At the end of reaction, 150 mL of water was added
to quench reaction, and products were extracted with ethyl
acetate (3 x 150 mL). The combined organic layers were
washed with water, dried by the anhydrous sodium sulfate
and filtered to obtain the mixture which was purified by
column chromatography to give mono-substituted
compounds 5a-5k and di-substituted compounds 6a-6k.
3159, 1661, 162, 1512, 1361 cm-1; HRMS (ESI): m/z calcd.
for C10H9ClN5O3S2: 330.0131 [M-H]+; found: 330.0137.
5-N-[2-(N-(4-bromophenyl)acetamide)]-5-amino-1,3,4-
thiadiazole-2-sulfonamide (5d): White solid; yield: 22%;
1
purity (HPLC): 99.62%; m.p. 198-200; H NMR (400 MHz,
DMSO-d6) δ 10.18 (s, 1H, -CONH), 8.80 (s, 1H, -NH-), 7.89
(s, 2H, -SO2NH2-), 7.60-7.39 (m, 4H, Ar-H), 3.90 (s, 2H, -
CH2-); 13C NMR (101 MHz, DMSO-d6) δ 172.46, 166.76,
155.71, 138.45, 132.08, 121.58, 115.52, 46.30; IR (KBr):
3435, 3333, 3269, 3193, 1675, 1606, 1591, 1388 cm-1;
HRMS (ESI): m/z calcd. for C10H9BrN5O3S2: 389.9330 [M-H]+;
found: 389.9339.
5-N-[2-(N-(4-chlorophenyl)acetamide)]-5-amino-1,3,4-
thiadiazole-2-sulfonamide (5a): White solid; yield: 21%;
5-N-[2-(N-(4-trifluoromethyl
phenyl)acetamide)]-5-
1
purity (HPLC): 99.88%; m.p. 217-219; H NMR (400 MHz,
amino-1,3,4-thiadiazole-2-sulfonamide (5e): White solid;
1
DMSO-d6) δ 10.18 (s, 1H, -CONH-), 8.81 (s, 1H, -NH-), 7.90
(s, 2H, -SO2NH2-), 7.69-7.49 (m, 2H, Ar-H), 7.49-7.25 (m, 2H,
Ar-H), 3.91 (s, 2H, -CH2-); 13CNMR (101 MHz, DMSO-d6) δ
172.46, 166.74, 155.68, 138.04, 129.19, 127.46, 121.18,
46.26; IR (KBr): 3447, 3335, 3263, 3202, 1684, 1610, 1595,
1359 cm-1; HRMS (ESI): m/z calcd. for C10H9ClN5O3S2:
345.9835 [M-H]+; found: 345.9842.
yield:19%; purity (HPLC): 99.31%; m.p. 225-227; H NMR
(400 MHz, DMSO-d6) δ 10.41 (s, 1H, -CONH), 8.84 (s, 1H, -
NH-), 7.90 (s, 2H, -SO2NH2-), 7.75 (d, J = 8.6 Hz, 2H, Ar-H),
7.68 (d, J = 8.7 Hz, 2H, Ar-H), 3.96 (s, 2H, -CH2-); 13C NMR
(126 MHz, DMSO-d6) δ 172.48, 167.30, 155.72, 142.61,
126.61 (d, J = 3.8 Hz), 124.79 (d, J = 272.2 Hz), 123.90 (d, J
= 44.1 Hz), 119.56, 46.33; IR (KBr): 3440, 3367, 3263, 3129,
1688, 1612, 1528, 1330 cm-1; HRMS (ESI): m/z calcd. for
C11H9F3N5O3S2: 380.0099 [M-H]+; found: 380.0104.
5-N-[2-(N-(4-methoxyphenyl)acetamide)]-5-amino-1,3,4-
thiadiazole-2-sulfonamide (5b): White solid; yield: 23%;
1
purity (HPLC)：99.81%; m.p. 218-220; H NMR (400 MHz,
5-N-[2-(N-(p-methylphenyl)acetamide)]-5-amino-1,3,4-
thiadiazole-2-sulfonamide (5f): White solid; yield: 25%;
DMSO-d6) δ 9.89 (s, 1H, -CONH-), 8.75 (s, 1H, -NH-), 7.89
(s, 2H,-SO2NH2-), 7.47-7.38 (m, 2H, Ar-H), 6.96-6.80 (m, 2H,
Ar-H), 3.86 (s, 2H, -CH2-), 3.72 (s, 3H, -OCH3); 13C NMR
(101 MHz, DMSO-d6) δ 172.46, 165.99, 155.81, 155.76,
132.19, 121.26, 114.36, 55.63, 46.20,40.66; IR (KBr): 3420,
3314, 3270, 3142, 1682, 1602, 1575, 1361 cm-1; HRMS
(ESI): m/z calcd. for C11H12N5O4S2: 342.0331 [M-H]+; found:
342.0339.
1
purity (HPLC): 99.72%; m.p. 211-213; H NMR (400 MHz,
DMSO-d6) δ 9.94 (s, 1H, -CONH-), 8.75 (d, J = 5.8 Hz, 1H,-
NH-), 7.89 (s, 2H, -SO2NH2-), 7.47-7.33 (m, 2H, Ar-H), 7.23-
7.01 (m, 2H, Ar-H), 3.88 (d, J = 4.5 Hz, 2H, -CH2-), 2.25 (s,
3H, -CH3); 13C NMR (101 MHz, DMSO-d6) δ 172.46, 166.24,
155.76, 136.56, 132.85, 129.62, 119.69, 46.24, 20.92; IR
(KBr): 3433, 3359, 3275, 3120, 1684, 1610, 1514, 1363 cm-
1; HRMS (ESI): m/z calcd. for C11H12N5O3S2: 326.0382 [M-
H]+; found: 326.0390.
5-N-[2-(N-(4-fluorophenyl)acetamide)]-5-amino-1,3,4-
thiadiazole-2-sulfonamide (5c): White solid; yield: 20%;
1
5-N-[2-(N-(4-iodophenyl)acetamide)]-5-amino-1,3,4-
purity (HPLC): 98.93%; m.p. 203-205; H NMR (400 MHz,
thiadiazole-2-sulfonamide (5g): White solid; yield: 27%;
DMSO-d6) δ 10.10 (s, 1H, -CONH-), 8.79 (t, J = 6.0 Hz, 1H,
-NH-), 7.90 (s, 2H, -SO2NH2-), 7.71-7.46 (m, 2H, Ar-H), 7.24-
7.04 (m, 2H, Ar-H), 3.89 (d, J = 5.9 Hz, 2H, -CH2-); 13C NMR
(101 MHz, DMSO-d6) δ 172.46, 166.47, 158.55 (d, J = 239.9
Hz), 155.73, 135.46 (d, J = 2.4 Hz), 121.46 (d, J = 7.9 Hz),
115.83 (d, J = 22.3 Hz), 46.21; IR (KBr): 3430, 3335, 3283,
1
purity (HPLC)：99.86%; m.p. 228-230; H NMR (400 MHz,
DMSO-d6) δ 10.14 (s, 1H, -CONH), 8.79 (s, 1H, -NH-), 7.89
(s, 2H, -SO2NH2-), 7.77-7.56 (m, 2H, Ar-H), 7.50-7.30 (m, 2H,
Ar-H), 3.90 (s, 2H, -CH2-); 13C NMR (101 MHz, DMSO-d6) δ
9
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172.46, 166.75, 155.70, 138.91, 137.91, 121.84, 87.47,
46.33; IR (KBr): 3429, 3326, 3289, 3127, 1682, 1611, 1543,
1386 cm-1; HRMS (ESI): m/z calcd. for C10H9IN5O3S2:
437.9191 [M-H]+; found: 437.9201.
DMSO-d6) δ 9.83 (s, 1H, -CONH-), 8.80 (s, 1H, -NH-), 7.89
(s, 2H, -SO2NH2-), 7.86-7.76 (m, 1H, Ar-H), 7.34-7.19 (m, 1H,
Ar-H), 7.22-7.04 (m, 2H, Ar-H), 3.98 (s, 2H, -CH2-); 13C NMR
(101 MHz, DMSO-d6) δ 172.48, 167.14, 155.68, 153.93 (d, J
= 245.1 Hz), 126.11 (d, J = 11.6 Hz), 125.88 (d, J = 7.4 Hz),
124.87 (d, J = 3.4 Hz), 124.38, 115.98 (d, J = 19.3 Hz), 45.99;
IR (KBr): 3420, 3345, 3275, 3131, 1690, 1620, 1495, 1359
cm-1; HRMS (ESI): m/z calcd. for C10H9FN5O3S2:
330.0131[M-H]+; found: 330.0139.
5-N-[2-(N-(4-tert-butylphenyl)acetamide)]-5-amino-1,3,4-
thiadiazole-2-sulfonamide (5h): White solid; yield: 23%;
1
purity (HPLC)：99.78%; m.p. 201-203; H NMR (400 MHz,
DMSO-d6) δ 9.96 (s, 1H, -CONH-), 8.76 (s, 1H, -NH-), 7.89
(s, 2H, -SO2NH2-), 7.53-7.40 (m, 2H, Ar-H), 7.40-7.24 (m, 2H,
Ar-H), 3.88 (s, 2H, -CH2-), 1.25 (s, 9H, -CH3); 13C NMR (101
MHz, DMSO-d6) δ 172.45, 166.28, 155.76, 146.25, 136.48,
125.85, 119.50, 46.21, 34.49, 31.66; IR (KBr): 3431, 3353,
3270, 3143, 1679, 1606, 1514, 1380 cm-1; HRMS (ESI): m/z
calcd. for C14H18N5O3S2: 368.0851 [M-H]+; found: 368.0860.
5-N,N-bis[2-(N-(4-chlorophenyl)acetamide)]-5-amino-
1,3,4-thiadiazole-2-sulfonamide (6a): White solid; yield:
65%; purity (HPLC): 98%; m.p. 216-218; 1H NMR (500 MHz,
DMSO-d6) δ 10.60 (s, 2H, -CONH-), 8.06 (s, 2H, -SO2NH2),
7.62 (d, J = 8.1 Hz, 4H, Ar-H), 7.41 (d, J = 8.3 Hz, 4H, Ar-H),
4.32 (s, 4H, -CH2-); 13C NMR (126 MHz, DMSO-d6) δ 172.81,
167.40, 153.30, 137.86, 129.31, 127.75, 121.26,52.98; IR
(KBr): 3412, 3290, 3186, 1677, 1604, 1547, 1367 cm-1;
HRMS (ESI): m/z calcd. for C18H15Cl2N6O4S2: 512.9973 [M-
H]+; found: 512.9983.
5-N-[2-(N-phenylacetamide)]-5-amino-1,3,4-thiadiazole-
2-sulfonamide (5i): White solid; yield: 22%; purity(HPLC):
97.61%; m.p. 185-187; 1H NMR (400 MHz, DMSO-d6) δ
10.03 (s, 1H, -CONH-), 8.78 (s, 1H, -NH-), 7.90 (s, 2H, -
SO2NH2-), 7.66-7.47 (m, 2H, Ar-H), 7.31 (t, J = 7.9 Hz, 2H,
Ar-H), 7.06 (t, J = 7.3 Hz, 1H, Ar-H), 3.91 (s, 2H, -CH2-); 13
C
5-N,N-bis[2-(N-(4-methoxyphenyl)acetamide)]-5-amino-
1,3,4-thiadiazole-2-sulfonamide (6b): White solid; yield:
NMR (101 MHz, DMSO-d6) δ 172.46, 166.53, 155.79, 139.07,
129.26, 123.93, 119.67, 46.28; IR (KBr): 3432, 3351, 3277,
3128, 1684, 1606, 1512, 1359 cm-1; HRMS (ESI): m/z calcd.
for C10H10N5O3S2: 312.0225[M-H]+; found: 312.0234.
1
67%; purity (HPLC): 95.17%; m.p. 233-235; H NMR (400
MHz, DMSO-d6) δ 10.47 (s, 2H, -CONH-), 8.07 (s, 2H, -
SO2NH2), 7.53 (d, J = 8.6 Hz, 4H, Ar-H), 6.93 (d, J = 8.7 Hz,
4H, Ar-H), 4.28 (s, 4H, -CH2-), 3.74 (s, 3H，-OCH3); 13C
NMR (126 MHz, DMSO-d6) δ 172.82, 166.80, 156.09, 153.32,
132.09, 121.35, 114.54, 55.71, 53.42; IR (KBr): 3396, 3292,
3122 ,1673, 1620, 1512, 1357 cm-1; HRMS (ESI): m/z calcd.
for C20H21N6O6S2: 505.0964 [M-H]+; found: 505.0974.
5-N-[2-(N-(3-fluorophenyl)acetamide)]-5-amino-1,3,4-
thiadiazole-2-sulfonamide (5j): White solid; yield: 23%;
1
purity (HPLC)：99.9%; m.p. 188-190; H NMR (400 MHz,
DMSO-d6) δ 10.26 (s, 1H, -CONH-), 8.82 (s, 1H, -NH-), 7.90
(s, 2H,-SO2NH2-), 7.52 (dt, J = 11.6, 2.3 Hz, 1H, Ar-H), 7.35
(td, J = 8.2, 6.7 Hz, 1H, Ar-H), 7.25 (dt, J = 8.4, 1.3 Hz, 1H,
Ar-H), 7.01 – 6.78 (m, 1H, Ar-H), 3.92 (s, 2H, -CH2-); 13C
NMR (101 MHz, DMSO-d6) δ 172.48, 166.97, 162.58 (d, J =
241.4 Hz), 155.71, 140.77 (d, J = 11.0 Hz), 130.94 (d, J = 9.5
Hz), 115.40 (d, J = 2.3 Hz), 110.42 (d, J = 21.1 Hz), 106.47
(d, J = 26.3 Hz), 46.29; IR (KBr): 3440, 3351, 3273, 3120,
1688, 1616, 1516, 1357 cm-1; HRMS (ESI): m/z calcd. for
C10H9FN5O3S2: 330.0131[M-H]+; found: 330.0140.
5-N,N-bis[2-(N-(4-fluorophenyl)acetamide)]-5-amino-
1,3,4-thiadiazole-2-sulfonamide (6c): White solid; yield:
1
60%; purity (HPLC): 97.66%; m.p. 216-218; H NMR (500
MHz, DMSO-d6) δ 10.57 (s, 2H, -CONH-), 8.07 (s, 2H, -
SO2NH2-), 7.62 (dd, J = 8.8, 4.9 Hz, 4H, Ar-H), 7.19 (t, J =
8.7 Hz, 4H, Ar-H), 4.31 (s, 4H, -CH2-); 13C NMR (126 MHz,
DMSO-d6) δ 172.81, 167.21, 158.70 (d, J = 240.2 Hz),
153.24, 135.31(d, J = 2.4 Hz), 121.48 (d, J = 7.8 Hz), 115.99
(d, J = 22.3 Hz), 53.08; IR (KBr): 3349, 3273, 3161, 1663,
1624, 1510, 1373 cm-1; HRMS (ESI): m/z calcd. for
C18H15F2N6O4S2: 481.0564 [M-H]+; found: 481.0574.
5-N-[2-(N-(2-fluorophenyl)acetamide)]-5-amino-1,3,4-
thiadiazole-2-sulfonamide (5k): White solid;yield: 22%;
1
purity (HPLC)：99.75%; m.p. 185-187; H NMR (400 MHz,
10
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5-N,N-bis[2-(N-(4-bromophenyl)acetamide)]-5-amino-
1,3,4-thiadiazole-2-sulfonamide (6h): White solid; yield:
1
1,3,4-thiadiazole-2-sulfonamide (6d): White solid; yield:
67%; purity (HPLC): 99.17%; m.p. 258-260; H NMR (500
1
MHz, DMSO-d6) δ 10.55 (s, 2H, -CONH-), 8.09 (s, 2H, -
SO2NH2-), 7.53 (d, J = 8.0 Hz, 4H, Ar-H), 7.36 (d, J = 8.3 Hz,
4H, Ar-H), 4.29 (s, 4H, -CH2-), 1.27 (s,18H, -CH3); 13C NMR
(126 MHz, DMSO-d6) δ 172.81, 167.16, 153.14, 146.54,
136.36, 125.99, 119.50, 53.42, 34.54, 31.66; IR (KBr): 3422,
3361, 3130, 1677, 1608, 1549, 1351 cm-1; HRMS (ESI): m/z
calcd. for C26H33N6O4S2: 557.2005 [M-H]+; found: 557.2015.
68%; purity (HPLC)：97.61%; m.p. 234-236; H NMR (500
MHz, DMSO-d6) δ 10.58 (s, 2H, -CONH-), 8.06 (s,2H, -
SO2NH2-), 7.55 (q, J = 8.8 Hz, 8H, Ar-H), 4.31 (s, 4H, -CH2-);
13C NMR (126 MHz, DMSO-d6) δ 173.03, 167.40, 153.84,
137.87, 132.07, 122.28, 116.30, 53.43; IR (KBr): 3439, 3298,
3179, 1669, 1610, 1547, 1369 cm-1; HRMS (ESI): m/z calcd.
for C18H15Br2N6O4S2: 602.8942 [M-H]+; found: 602.8953.
5-N,N-bis[2-(N-phenyl)acetamide)]-5-amino-1,3,4-
5-N,N-bis[2-(N-(4-trifluoromethyl phenyl)acetamide)]-5-
thiadiazole-2-sulfonamide (6i): White solid; yield: 66%;
amino-1,3,4-thiadiazole-2-sulfonamide (6e): White solid;
1
1
purity(HPLC): 98.42%; m.p. 226-228; H NMR (500 MHz,
yield: 70%; purity(HPLC)：97.7%; m.p. 222-224; H NMR
DMSO-d6) δ 10.55 (s, 2H, -CONH-), 8.08 (s, 2H, -SO2NH2-),
7.62 (d, J = 7.9 Hz, 4H, Ar-H), 7.35 (t, J = 7.7 Hz, 4H, Ar-H),
7.10 (t, J = 7.4 Hz, 2H, Ar-H), 4.32 (s, 4H, -CH2-); 13C NMR
(126 MHz, DMSO-d6) δ 172.82, 167.32, 153.24, 138.94,
129.39, 124.19, 119.69, 53.30; IR (KBr): 3381, 3318, 3108,
1665, 1602, 1551, 1375 cm-1; HRMS (ESI): m/z calcd. for
C18H17N6O4S2: 445.0753 [M-H]+; found: 445.0762.
(500 MHz, DMSO-d6) δ 10.77 (s, 2H, -CONH-), 8.06 (s, 2H,
-SO2NH2-), 7.88-7.51 (m, 8H, Ar-H), 4.38 (s, 4H, -CH2-); 13
C
NMR (126 MHz, DMSO-d6) δ 172.81, 167.87, 153.37, 142.47,
126.73 (q, J = 3.8 Hz), 124.78 (q, J = 272.16 Hz), 124.15 (q,
J = 31.5 Hz), 119.64, 52.84; IR (KBr): 3477, 3326, 3185,
1677, 1610, 1551, 1324 cm-1; HRMS (ESI): m/z calcd. for
C20H15F6N6O4S2: 581.0500 [M-H]+; found: 581.0511.
5-N,N-bis[2-(N-(3-fluorophenyl)acetamide)]-5-amino-
5-N,N-bis[2-(N-(p-methylphenyl)acetamide)]-5-amino-
1,3,4-thiadiazole-2-sulfonamide (6f): White solid; yield:
67%; purity (HPLC)：97%; m.p. 225-227; 1H NMR (500 MHz,
DMSO-d6) δ 10.48 (s, 2H, -CONH-), 8.08 (s, 2H, -SO2NH2-),
7.50 (d, J = 8.0 Hz, 4H, Ar-H), 7.15 (d, J = 8.1 Hz, 4H, -H),
4.29 (s, 4H, -CH2-), 2.27 (s, 6H, -CH3); 13C NMR (126 MHz,
DMSO-d6) δ 172.81, 167.07, 153.20, 136.43, 133.15, 129.75,
119.70, 53.33, 20.94; IR (KBr): 3378, 3294, 3190, 1673,
1614, 1553, 1363 cm-1; HRMS (ESI): m/z calcd. for
C20H21N6O4S2: 473.1066 [M-H]+; found: 473.1075.
1,3,4-thiadiazole-2-sulfonamide (6j): White solid; yield:
1
66%; purity (HPLC)：96.50%; m.p. 230-232; H NMR (400
MHz, DMSO-d6) δ 10.24 (s, 2H, -CONH-), 8.05 (s, 2H, -
SO2NH2-), 7.57 (dt, J = 11.5, 2.3 Hz, 2H, Ar-H), 7.39 (td, J =
8.2, 6.6 Hz, 2H, Ar-H), 7.30 (dt, J = 8.5, 1.3 Hz, 2H, Ar-H),
7.02-6.82 (m, 2H, Ar-H), 4.33 (s, 4H, -CH2-); 13C NMR (101
MHz, DMSO-d6) δ 172.83, 167.59, 162.61 (d, J = 241.6 Hz),
153.33, 140.60 (d, J = 11.0 Hz), 131.10 (d, J = 9.5 Hz),
115.44(d, J = 2.3 Hz), 110.68 (d, J = 21.1 Hz), 106.51 (d, J =
26.4 Hz), 52.86; IR (KBr): 3392, 3271, 3146, 1679, 1614,
1563, 1363 cm-1; HRMS (ESI): m/z calcd. for
C18H15F2N6O4S2: 481.0564 [M-H]+; found: 481.0575.
5-N,N-bis[2-(N-(4-iodophenyl)acetamide)]-5-amino-1,3,4-
thiadiazole-2-sulfonamide (6g): White solid; yield: 60%;
1
purity (HPLC): 99.77%; m.p. 211-213; H NMR (500 MHz,
5-N,N-bis[2-(N-(2-fluorophenyl)acetamide)]-5-amino-
DMSO-d6) δ 10.55 (s, 2H, -CONH-), 8.05 (s, 2H, -SO2NH2-),
7.68 (d, J = 8.4 Hz, 4H, Ar-H), 7.43 (d, J = 8.4 Hz, 4H, Ar-H),
4.30 (s, 4H, -CH2-); 13C NMR (126 MHz, DMSO-d6) δ 172.80,
167.42, 153.29, 138.73, 138.04, 121.89, 87.79, 53.02; IR
(KBr): 3435, 3298, 3175, 1677, 1608, 1539, 1369 cm-1;
HRMS (ESI): m/z calcd. for C18H15I2N6O4S2: 696.8686 [M-H]+;
found: 696.8696.
1,3,4-thiadiazole-2-sulfonamide
(6k):
White
solid;
1
yield:65%; purity (HPLC): 97.99%; m.p. 230-232; H NMR
(400 MHz, DMSO-d6) δ 10.24 (s, 2H, -CONH-), 8.05 (s, 2H,
-SO2NH2-), 7.92 (td, J = 7.8, 3.5 Hz, 2H, Ar-H), 7.28 (ddd, J
= 11.1, 6.8, 3.1 Hz, 2H, Ar-H), 7.18 (dq, J = 6.6, 3.8, 3.2 Hz,
4H, Ar-H), 4.38 (s, 4H, -CH2-); 13C NMR (101 MHz, DMSO-
d6) δ 172.82, 167.68, 153.82 (d, J = 245.4 Hz), 153.28,
126.09 (d, J = 11.5 Hz), 125.94 (d, J = 7.3 Hz), 124.90 (d, J
5-N,N-bis[2-(N-(4-tert-butylphenyl)acetamide)]-5-amino-
11
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10.1002/cmdc.201900703
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FULL PAPER
= 3.3 Hz), 124.20, 116.04 (d, J = 19.2 Hz), 52.83; IR (KBr):
3461, 3267, 3137, 1675, 1622, 1547, 1373 cm-1; HRMS
(ESI): m/z calcd. for C18H15F2N6O4S2: 481.0564 [M-H]+;
found: 481.0574.
4 h at 37 °C in the presence of 5% CO2. The supernatant
was discarded, 100 μL of DMSO was added to the wells of
96-well plates, and the solutions were mixed thoroughly. The
absorbance was recorded at 570 nm using a microplate
reader. The experiments were repeated at least 3 times.
Biological assays
Cytotoxicity assay: The cells (HEK293, LO2 and PC12)
were respectively seeded into 96-well plates (5×103
cells/well) and cultured at 37℃ and 5% CO2 for 24 h. The
Carbonic anhydrase inhibition assay: According to
previously reported method,[34] 4-NPA can be converted into
4-nitrophenol with catalysed by carbonic anhydrase, and the
reaction rate was determined by the spectrophotometry at
405 nm with an enzyme labelling instrument.[42,43] Human
carbonic anhydrases I, II and IX were prepared in 250 ng/μL
reserve solutions for the subsequent usage. Buffer was used
to dilute hCA I, hCA II and hCA IX protein reserve solutions
to 2.5 ng/μL, 5 ng/μL and 10 ng/μL, respectively, and they
were then added to 384-well reaction plates. The experiment
was divided into three groups: blank group, positive control
group (AZA) and sample group. All inhibitors were diluted
into seven different concentrations in a gradient and added
to 384-well reaction plates in triplicate to form enzyme
inhibitor (E-I) complexes; the complexes were incubated at
25℃ for 15 minutes. The substrate 4-NPA was added to the
E-I solution and the incubation continued for 1 h (CA I), 1 h
(CA II) and 4 h (CA IX), respectively. The absorbance of
solution was measured at 405 nm by microplate reader
(BioTek, Epoch, USA). IC50 value was obtained with
GraphPadPrism 5.0 software. The experiments were
repeated at least 3 times.
medium was removed, and 100 μL of new medium
containing different concentrations of compounds was added
and treated for 48 h. MTT (5 mg/mL, 10 μL) was added to
each well and treated for 4 h at 37℃ and 5% CO2. The
supernatant in each well was removed, and 100 μL of DMSO
were added to the wells of 96-well plates, and the solutions
were mixed thoroughly. The absorbance was recorded at
570 nm using a microplate reader. IC50 values of compounds
were calculated by GraphPad Prism 5.0 software. The
experiments were repeated at least 3 times.
Computational assays
Molecular docking: The structures of ligands were drawn
by ChembioDraw Ultra 14.0 software and saved in SDF
format. The crystal structure of human CA I (PDB code:
5WLU),[37] human CA II (PDB code: 5ULN)[38] and human CA
IX (PDB code: 5JN3)[39] were derived from RCSB PDB data
bank, with 1.39 Å, 1.35 Å and 1.60 Å resolution. The proteins
were treated with the process where all water was removed
and hydrogen bonds were added. PyMol [The PyMol
Molecular Graphics System, version 1.1 Schrödinger LLC]
software was used to dock protein and compounds and to
find the best docking posture for CA II and compound 5c.
Cell culture: PC12 cells were cultured in DMEM containing
5% FBS, 5% HS, 100 U/mL penicillin and 100 μg/mL
streptomycin at 37℃ and 5% CO2. HEK293 and LO2 cells
were cultured in DMEM containing 10% FBS, 100 U/mL
penicillin and 100 μg/mL streptomycin at 37℃ and 5% CO2.
When the cells were 80%-90% confluent, they were digested
with 0.25% trypsin -0.02% EDTA and passaged.
Prediction of ADME Properties: %ABS (percentage
absorption), MW (molecular weight), miLog P (logarithm of
partition coefficient of compound between n-octanol and
water), TPSA (topological polar surface area), n-ROTB
(number of rotatable bonds), n-ON acceptors (number of
hydrogen bond acceptors), and n-OHNH donors (number of
hydrogen bond donors) were calculated by Molinspiration
Online Property Calculation Toolkit (Molinspiration,2015).[44]
Absorption (% ABS) was calculated by using the equation %
ABS = 109-(0.345 x TPSA).[45,46]
Cell viability assay: Cell viability was measured by MTT
assay. Briefly, the PC12 cells were seeded in 96-well plates
(1.5×104 cells/well) and cultured for 24 h. The cells were then
pre-treated with the indicated concentrations of target
compounds for 2 h, and incubated with 650 µM SNP for 24
h. 10 μL of 5 mg/mL MTT were added to pre-treated cells for
12
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[12] A. Nocentini, P. Gratteri, C. T. Supuran. Chem Eur J.
2019, 25, 1188-1192.
Acknowledgements
This research was financially supported by the Science and
Technology Planning Program of Guangzhou ( Grant No.
201704020146).
[13] E. Masini, F. Carta, A. Scozzafava, C. T. Supuran.
Expert Opinion Ther Pat. 2013, 26, 705-716.
[14] T. V. Wani, S. Bua, P. S. Khude, A. H. Chowdhary, C.
T. Supuran & M. P. Toraskar. J Enzyme Inhib Med
Chem. 2018, 33, 962-971.
Conflict of interest
The authors declare no conflict of interest.
[15] C. B. Mishra, S. Kumari, A. Angeli, S. Bua, M.
Buonanno, S. M. Monti, M. Tiwari, C.T. Supuran. Eur J
Med Chem. 2018, 156, 430-443.
Keywords: Acetazolamide derivatives • carbonic anhydrase
II (CA II) • inhibitor • neuroprotection
[16] S. Schwarz, S. Sommerwerk, S. D. Lucas, L. Heller, R.
Csuk. Eur J Med Chem. 2014, 86, 95-102.
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Entry for the Table of Contents
Compound 5c was the most effective selective carbonic anhydrase II inhibitor (IC50=16.7 nM, CAI/CAII=54.3), which was
comparable to the inhibitory effect of acetazolamide. Moreover, the selectivity of compound 5c was better than that of
acetazolamide (IC50=12.0 nM, CAI/CAII=20.8). Compound 5c exhibited a better protective effect for PC12 cells and showed
less cytotoxicity than that of acetazolamide.
15
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